The mechanism and control of lipoamide dehydrogenase and two related pyridine nucleotide-disulfide oxidoreductases, glutathione reductase and thioredoxin reductase are being studied. The reactions catalyzed by each of these flavoenzymes involve the transfer of two electrons via the FAD and the disulfide of a cystine residue. Peptides containing the active site cystines have been sequenced an they show structural similarities (tight loops) analogous to the mechanistic similarities in this group. A high degree of homology exists in this region of the protein between lipoamide dehydrogenase and glutathione reductase but these show no homology to thioredoxin reductase. There is a base at the active site of lipoamide dehydrogenase and glutathione reductase which functions in the deprotonation of the dithiol substrate (lipoamide dehydrogenase) or in the protonation of the reduced glutathione. This base also interacts with each of the active center thiols increasing their nucleophilicity at appropriate points in catalysis. The distinct functions of each active site have been determined: the one nearer the amino-terminus of the protein interacts with the dithiol substrate via thioldisulfide interchange and the one nearer the carboxyl-terminus interacts with the FAD at the C(4a) position. The pyridine nucleotide half reaction of lipoamide dehydrogenase is being studied in the hope of identifying intermediates. The studies on lipoamide dehydrogenase and glutathione reductase are being extended to thioredoxin reductase.